CN112852702A

CN112852702A - Method for synthesizing tagatose from high-concentration lactose under catalysis of recombinant bacillus subtilis

Info

Publication number: CN112852702A
Application number: CN202110280304.8A
Authority: CN
Inventors: 饶志明; 李志月; 张显; 杨套伟; 徐美娟; 邵明龙
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2021-05-28
Anticipated expiration: 2041-03-16
Also published as: CN112852702B

Abstract

The invention discloses a method for synthesizing tagatose from high-concentration lactose by catalyzing recombinant bacillus subtilis, and belongs to the technical field of enzyme engineering and microbial engineering. The food-grade safe recombinant strain provided by the invention can realize that the genetic stability of plasmid in host cells is maintained without adding antibiotics in the cell culture process, tagatose is safely and efficiently synthesized by a one-step biological method of whole-cell catalysis lactose, the maximum molar conversion rate of 57.23% is obtained by controlling the pH of a reaction system when 100g/L of substrate lactose is added, and the highest yield of tagatose is 96.76g/L when the substrate loading capacity is increased to 500g/L through the optimization of a whole-cell catalysis process. The method reduces the production cost of the tagatose, improves the concentration of the substrate, improves the safety level of the tagatose applied to the food field, realizes the high-efficiency synthesis of the tagatose, and lays a solid research foundation for the safe food industrial production of the tagatose.

Description

Method for synthesizing tagatose from high-concentration lactose under catalysis of recombinant bacillus subtilis

Technical Field

The invention relates to a method for synthesizing tagatose from high-concentration lactose by catalyzing recombinant bacillus subtilis, belonging to the technical field of enzyme engineering and microbial engineering.

Background

Tagatose, a rare sugar from natural sources, an epimer of fructose, has sweetness similar to that of sucrose, but has the heat of only 1/3 of the sucrose, has various physiological effects of inhibiting hyperglycemia, improving intestinal flora, avoiding decayed teeth and the like, becomes a good sweet substitute for development of health products and upgrading of foods, and is widely applied to the fields of foods, medicines, cosmetics and the like. D-tagatose was recognized as safe food (GRAS) by the U.S. Food and Drug Administration (FDA) in 2001, and thereafter, in 2004, tagatose was approved as safe food by the Joint Experts Committee for Food Additives (JECFA) of the world health organization. The global artificial sweetener market in 2016 is about $ 32 billion, and in the next few years, the tagatose market has expanded.

The tagatose can be produced by chemical and biological methods. The tagatose produced by the chemical catalysis method of the calcium catalyst and the strong acid has the defects of generation of byproducts and chemical wastes and the like, and the biological method utilizes arabinose isomerase to catalyze galactose to isomerize and produce the tagatose and has the characteristics of mild reaction conditions, few byproducts, environmental friendliness and the like. However, arabinose isomerase has problems of low kinetics, low thermostability and low equilibrium constant (galactose/tagatose ratio is about 7: 3) which are unfavorable to galactose substrate, so that the industrial production of tagatose is hindered by the problems of high cost and low conversion rate in tagatose production. In the method for producing tagatose by a biological enzyme method disclosed by Zhang Houcheng et al in 2011, the tagatose is synthesized by constructing an arabinose isomerase enzyme method efficiently expressed by an escherichia coli engineering strain through catalysis, the conversion rate reaches 39.5%, and a substrate is galactose. On the other hand, the antibiotic resistance gene carried by the genetically engineered bacteria and the antibiotic added in the culture process all bring threat to food safety, and the safe and high yield of tagatose becomes a problem to be solved urgently.

Disclosure of Invention

The invention provides a method for synthesizing tagatose by catalyzing lactose through food-grade safe recombinant bacillus subtilis whole-cell combination. In order to reduce the problem of high cost caused by unfavorable kinetics of arabinose isomerase to galactose, the galactose is produced by hydrolyzing relatively cheap lactose with beta-galactosidase so as to reduce the substrate cost, GARS strain-bacillus subtilis with mature fermentation technology is selected as a host to respectively express beta-galactosidase and arabinose isomerase from escherichia coli, and alanine racemase gene is used as a screening marker to construct food-grade safe recombinant bacillus subtilis. Compared with pure enzyme conversion, the whole-cell conversion has the characteristics of environmental disturbance resistance and lower effective enzyme cost, the stability duration of the intracellular enzyme is prolonged to a certain extent, and the isomerization reaction catalyzed by the arabinose isomerase is shifted to the direction of the tagatose product more by optimizing the whole-cell conversion condition process, so that the aim of efficiently synthesizing the tagatose is fulfilled.

The first object of the present invention is to provide a composition comprising a recombinant bacterium expressing β -galactosidase and a recombinant bacterium expressing arabinose isomerase.

In one embodiment of the invention, the recombinant bacterium expresses the alanine racemase gene alrA.

In one embodiment of the invention, the beta-galactosidase and the arabinose isomerase are both derived from escherichia coli, and the nucleotide sequences are shown as SEQ ID No.2 and SEQ ID No. 3.

In one embodiment of the invention, the nucleotide sequence of the alanine racemase gene alrA is shown in SEQ ID NO. 1.

In one embodiment of the invention, the recombinant bacterium takes bacillus subtilis168 as a host and pMA5 as an expression vector.

In one embodiment of the invention, the bacillus subtilis168 has removed the alanine racemase gene alrA.

In one embodiment of the invention, the expression vector pMA5 has the kanamycin and bleomycin resistance genes removed.

In one embodiment of the invention, the nucleotide sequence of the kanamycin resistance gene is shown as SEQ ID No. 4; the nucleotide sequence of the bleomycin resistance gene is shown as SEQ ID NO. 5.

The second purpose of the invention is to provide a construction method of the recombinant bacillus subtilis, which comprises the following specific steps:

1) knocking out an alanine racemase alrA gene on the bacillus subtilis B.subtilis168 by using a cre/lox specific recombination system to obtain a D-alanine auxotrophic strain B.subtilis168D 1;

2) substituting the alanine racemase alrA gene for kanamycin and bleomycin resistance genes of an expression vector pMA5 to obtain an expression vector pMA5 a;

3) respectively constructing beta-galactosidase and arabinose isomerase genes derived from escherichia coli to an expression vector pMA5a to obtain plasmids pMA5a-lacZ and pMA5 a-araA;

4) plasmids pMA5a-lacZ and pMA5a-araA were introduced into D-alanine auxotrophic strain B.subtiliss 168D1 to obtain recombinant Bacillus subtilis B.subtiliss 168D1/pMA5a-lacZ and B.subtiliss D1/pMA5 3616835-araA, respectively.

The third purpose of the invention is to provide a safe and efficient tagatose synthesis method, which comprises the following steps: respectively inoculating the recombinant bacteria into an LB culture medium, culturing for 10-12h at 35-39 ℃ and 200-220 r/min, then inoculating into a TB culture medium according to the inoculum size of 1% (v/v), culturing for 20-30 h at 30-39 ℃ and 200-220 r/min, then collecting bacteria liquid, centrifugally collecting bacteria, washing cells by using physiological saline, suspending in 0.2mol/L Na₂HPO₄-obtaining a suspension in a buffer of citric acid; taking lactose as a substrate, adding recombinant bacteria B.subtilis168D1/pMA5a-lacZ and B.subtilis168D1/pMA5a-araA suspension, and reacting for 70-90 h.

In one embodiment of the invention, the process is a reaction at a pH of 8.0 to 9.0.

In one embodiment of the invention, the process is carried out at a temperature of from 45 to 55 ℃.

In one embodiment of the invention, the cell number ratio of the recombinant bacillus subtilis expressing beta-galactosidase to the recombinant bacillus subtilis expressing arabinose isomerase in the reaction system is1 (15-20).

In one embodiment of the invention, 2-5 mmol/L Mn is added into the reaction system of the method²⁺。

In one embodiment of the invention, 0.1 percent TritonX-100 of permeabilizing agent is added into a reaction system of the method.

In one embodiment of the present invention, the concentration of the bacterial suspension in the reaction system of the method is OD ₆₀₀40 to 50.

The invention also provides the application of the food-grade safe recombinant bacillus subtilis or the gene or the recombinant plasmid or the host cell or the preparation method in preparing food, medicines, health-care products or cosmetics.

Has the advantages that: (1) the invention provides a biocatalysis method for synthesizing tagatose by a one-step method, which has the characteristics of safety and high efficiency.

(2) The invention constructs food-grade recombinant bacillus subtilis chassis cells which keep plasmid stability without adding antibiotics in the culture process by utilizing a cre/lox specific recombination system and a gene recombination technology, and provides a safe cell support platform for the synthesis of a plurality of food-grade products.

(3) The invention expresses beta-galactosyl glycase and arabinose isomerase originated from escherichia coli in a recognized food safety strain B.subtilis168 engineering modified strain, researches the optimum condition of synthesizing tagatose by one-step method through converting lactose by whole cells of mixed bacterial suspension, obtains the maximum molar conversion rate of 57.23 percent when 100g/L of substrate lactose is added, obtains the highest yield of the tagatose of 96.76g/L when the substrate loading capacity is increased to 500g/L, reduces the cost of the tagatose synthesis process, improves the safety level of the tagatose applied to the food field, realizes the high-efficiency synthesis of the tagatose, and lays a good research foundation for the industrial production of the tagatose.

Drawings

FIG. 1: constructing a schematic diagram of the food-grade recombinant bacillus subtilis.

FIG. 2: a bacillus subtilis-verified nucleic acid electropherogram for knocking out alanine racemase gene alrA; wherein M is 10000bp marker; 1: a subtilis168 PCR product; 2: subtilis D1 (Zeo)^r) PCR products; 3: subtilis168D1 (Kan)^r) PCR products; 4: subtilis168D1 PCR product.

FIG. 3: recombinant plasmid verified nucleic acid gel electrophoresis; wherein M is 10000bp marker; 1 is an alrA gene on a recombinant plasmid pMA5 a; 2 is the araA gene on the recombinant plasmid pMA5 a-araA; 3 is the lacZ gene on the recombinant plasmid pMA5 a-lacZ.

FIG. 4: SDS-PAGE analysis of beta-galactosidase and arabinose isomerase expression; wherein, M: protein marker, 1: crude enzyme solution (control) 2 of subtilis168D1/pMA5 a: subtilis168D1/pMA5a-lacZ crude enzyme solution, 3: sublis 168D1/pMA5a-lacZ wall-breaking precipitate, 4: crude enzyme solution of subtilis168D1/pMA5a-araA, 5: sublilis 168D1/pMA5a-lacZ wall-broken precipitate.

FIG. 5: optimization analysis of whole cell transformation conditions (mixing ratio of two strains, optimum pH, temperature, and metal ions Mn)²⁺Concentration, permeabilizer triton x-100 concentration, bacterial suspension concentration, etc.).

FIG. 6: and (4) synthetic analysis of tagatose.

Detailed Description

The invention will be further illustrated with reference to specific examples.

The pMA5, p7Z6, pTSC plasmid and B.subtilis168 referred to in the examples below were all purchased from Invitrogen.

The media involved in the following examples are as follows:

LB culture medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract) and 10g/L of sodium chloride (NaCl).

TB culture medium: tryptone (Tryptone)12g/L, Yeast extract (Yeast extract)24g/L, K g/L₂HPO₄·3H₂O 16.4g/L、KH₂PO₄2.3g/L, Glycine (Glycine)7.5g/L and glycerol (glycerol) 5 g/L.

The detection methods referred to in the following examples are as follows:

method for determination of substrates and products:

and (3) detecting the substrate and the product by adopting High Performance Liquid Chromatography (HPLC): a Carbomix-Ca-NP chromatographic column, an ultrapure water mobile phase, a flow rate of 0.6mL/min, a column temperature of 80 ℃, a differential detector, a detection temperature of 50 ℃ and a sample injection amount of 20 mu L.

The enzyme activity determination method of beta-galactosidase and arabinose isomerase comprises the following steps:

enzyme activity assay reaction system (1 mL): 100g/L substrate, 200. mu.L pure enzyme, Na₂HPO₄Citric acid buffer (0.2mol/L, pH 7.0), reacted at 37 ℃ for 20min, and placed on ice to terminate the reaction. Protein concentration was determined by the Bradford method.

The enzyme activity of beta-galactosidase is defined by the amount of glucose produced in the enzyme-catalyzed reaction and the enzyme activity of arabinose isomerase is defined by the amount of tagatose produced.

Beta-galactosidase enzyme activity is defined as: under standard conditions (pH 7.0, 37 ℃), lactose is used as a substrate, and the enzyme amount required for catalyzing and generating 1 mu mol of product glucose per minute is one enzyme activity unit.

Arabinose isomerase enzyme activity is defined as: under standard conditions (pH 7.0 and 37 ℃), galactose is used as a substrate, and the enzyme amount required for catalyzing and generating 1 mu mol of tagatose product per minute is one enzyme activity unit.

Example 1: construction of alanine auxotroph bacillus subtilis (B.subtilis)168D1 and alanine racemase gene complementation bacillus subtilis B.subtilis168D1/pMA5a

(1) Construction and verification of alanine auxotroph bacillus subtilis168D1

1) Obtaining 800bp sequences of alanine racemase alrA and alrB in bacillus subtilis (B.subtilis)168 from NCBI as homology arms, carrying out plasmid map analysis by P7Z6, designing primers, and respectively amplifying the 800bp sequences of alanine racemase alrA and lox71-zeo on P7Z6 plasmid in the bacillus subtilis168 by using the primers P1/P2, P3/P4 and P5/P6^r-lox66 cassette sequence, using primers P7/P8, P9/P10 to amplify separately the Bacillus subtilis budIn bacillus 168, the sequence of 800bp is respectively before and after the alanine racemase alrB. The 800bp sequences of the alanine racemase AlrA and lox71-zeo on the P7Z6 plasmid were each sequenced by fusion PCR using primers P1/P4^r-the lox66 cassette sequence is ligated into a whole gene fragment a; the 800bp sequences of alanine racemase AlrB and before and after each were fused by PCR using primers P7/P10 with lox71-zeo on the P7Z6 plasmid^rThe lox66 cassette sequence is ligated into a whole gene fragment B.

2) The gene fragments A and B are respectively introduced into the competence of the bacillus subtilis168 by a chemical transformation method to obtain transformants, and LB solid plates containing 30mg/L of bleomycin are coated for screening. Due to the existence of the homology arms, homologous recombination occurs, and bacillus subtilis168 with the alanine racemase alrA deleted and bacillus subtilis168 with the alanine racemase alrB deleted are obtained.

3) Introducing the temperature-sensitive plasmid pTSC with the kanamycin resistance gene into Bacillus subtilis168 with alanine racemase alrA knock-out, inducing by using IPTG, carrying out specific recombination on lox71 and lox66 sites under the action of cre recombinase encoded on the temperature-sensitive plasmid pTSC, deleting the zeo resistance gene to form lox72 double mutation sites, and obtaining a transformant without the zeo resistance gene. The transformant was spotted on LB plates supplemented with 100. mu.g/mL D-alanine, and cultured at 51 ℃ for 48 hours to eliminate the thermo-sensitive plasmid pTSC, to obtain antibody-free, plasmid-free alanine racemase gene knock-out bacteria B.subtilis168D1, as shown in FIG. 1. The construction method of the alanine racemase gene knock-out bacterium B.subtilis168 delta-alrB is the same as that of the strain B.subtilis168D 1.

And respectively inoculating alanine racemase gene knock-out bacteria B.subtilis168D1 and B.subtilis168 delta alrB on an unresponsive LB plate and an unresponsive LB plate added with 100 mu g/mL of D-alanine, culturing for 12h at 37 ℃, and obtaining the alanine auxotrophic bacillus subtilis which is successfully constructed and does not grow on the unresponsive LB plate but grows on the unresponsive LB plate added with the D-alanine. The results are shown in FIG. 2, which shows that the ALrA-knockout Bacillus subtilis modified strain is successfully constructed, and a D-alanine auxotroph strain B.subtilis168D1 is obtained.

(2) Construction of alanine racemase Gene complementation of Bacillus subtilis168D1/pMA5a

Amplifying an alanine racemase gene alrA on a bacillus subtilis genome by using a primer P11/P12, reversely amplifying a plasmid pMA5 at two ends of kanamycin and bleomycin resistance genes on a pMA5 vector by using a primer P13/P14 to remove the kanamycin and bleomycin resistance genes, respectively carrying out agarose gel electrophoresis on a PCR product alanine racemase gene alrA gene fragment and a plasmid pMA5 which removes the kanamycin and bleomycin resistance genes, recovering and purifying gel, connecting the alrA gene fragment by using a homologous recombinase, and removing the kanamycin and bleomycin resistance gene plasmid pMA5 to obtain a recombinant vector pMA5a (figure 3); introducing the recombinant plasmid pMA5a into Escherichia coli JM109 through chemical transformation to obtain a transformant, coating the transformant on an LB solid culture medium, standing and culturing at 37 ℃ for 10-12h, carrying out colony PCR by using a primer P1/P4, selecting a positive clone, inoculating the positive clone into 10mL of LB culture medium, culturing at 37 ℃ at 180rpm/min for 12h, and extracting a plasmid according to a bacterial plasmid extraction kit; and introducing the amplified plasmid into bacillus subtilis168D1 by a chemical transformation mode to obtain a transformant, coating the transformant on an LB solid plate, performing static culture at 37 ℃ for 10-12h, performing colony PCR verification by using a primer P1/P4, selecting a positive clone, inoculating the positive clone into 10mL of LB medium at 37 ℃, and performing culture at 180rpm for 12h to obtain a recombinant strain B.subtilis168D1/pMA5 a.

TABLE 1 primer Table

TABLE 2 PCR reaction System

And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 30s (annealing temperature determined by primer), extension at 72 ℃ (extension time determined by amplified fragment length, 30 s. kb^-1) 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Homologous recombination: commercial homologous recombination kits were used, purchased from Nanjing Novozam, Inc.

And (3) glue recovery: the glue recovery kit is purchased from Shanghai Czeri bioengineering, Inc.

Example 2: heterologous expression of E.coli-derived beta-galactosidase and arabinose isomerase in B.subtiliss 168D1/pMA5a, respectively

(1) Construction and transformation of Gene expression vectors

The primers P15/P16 and P17/P18 were used to amplify beta-galactosidase and arabinose isomerase genes, respectively, from the E.coli genome by PCR, the plasmid pMA5a obtained in example 1 was digested simultaneously with Nde I and Mlu I fast-cutting enzymes, the PCR products beta-galactosidase and arabinose isomerase gene fragments were ligated to the digested products, respectively, by homologous recombination, and introduced into B.subtilis168D1, to obtain recombinant strains B.subtiliss 168D1/pMA5a-lacZ and B.subtiliss 168D1/pMA5 a-araA.

(2) Expression and enzyme activity determination of beta-galactosidase (lacZ) and arabinose isomerase (L-AI)

The recombinant Bacillus subtilis B.subtiliss 168D1/pMA5a-lacZ and B.subtiliss 168D1/pMA5a-araA obtained in the step (1) of the example 2 are respectively cultured in 10mL LB culture medium at 37 ℃ and 180r/min for 12h, then transferred to 50mL TB culture medium according to the inoculum size of 1% (v/v), cultured at 37 ℃ and 180rpm for 24h for expressing beta-galactosidase and arabinose isomerase, collected bacterial liquid, centrifuged to collect cells, washed twice with physiological salt and suspended in 5mL Na₂HPO₄Citric acid buffer (0.2mol/L, pH 8.0), adding 30 μ L lysozyme (200mg/mL) to the cells, standing on ice for 5h, ultrasonicating (400w break 1s stop 3s, 30min), centrifuging at 10,000r/min for 20min to obtain cell-broken supernatant, cell-broken precipitate, and filtering the supernatant with 0.22 μm filter membrane to obtain crude enzyme solution.

SDS-PAGE analysis is respectively carried out on the wall-broken supernatant and the wall-broken sediment, as shown in figure 4, the beta-galactosidase and the arabinose isomerase respectively have obvious bands near 110kDa and 55kDa, and heterologous expression is successfully realized. The enzyme activity data of beta-galactosidase and arabinose isomerase are shown in the following table 1 after enzyme activity determination:

table 2: beta-galactosidase lacZ and arabinose isomerase araA enzyme activities

Example 3: optimization analysis of Whole cell transformation conditions

(1) Subtiliss 168D1/pMA5 a-lacZ: B.Subtilis168D1/pMA5a-araA Whole cell transformation optimum cell number mixture ratio analysis

Controlling the amount of bacteria to be added OD₆₀₀All are OD₆₀₀Adjusting b.subtiliss 168d1/pMA5 a-lacZ: the ratio of the number of cells of subtilis168D1/pMA5a-araA was 1:1, 1:10, 1:15, 1:20, 1:30, respectively.

The reaction system is 5 mL: 0.2mol/L Na₂HPO₄Citric acid buffer (pH 8.0), 100g/L lactose, 5mmol/LMn²⁺0.1 percent of permeabilizing agent TritonX-100. After reacting for 12h at 50 ℃, the supernatant was collected by centrifugation, and the reaction was stopped by placing on ice, and after centrifugation, the amounts of the substrate and the product were analyzed by HPLC.

As shown in FIG. 5(A), when B.subtilis168D1/pMA5 a-lacZ: the highest yield of 16.06g/L was 1.5 times the yield of 1:1 for the subtiliss 168D1/pMA5a-araA whole cell transformation mix ratio of 1: 15.

(1) Optimum pH and temperature for whole cell transformation

Determination of the optimum pH: sublilis 168d1/pMA5 a-lacZ: the relative enzyme activities of beta-galactosidase and arabinose isomerase were measured by mixing subtiliss 168D1/pMA5a-araA at a cell number ratio of 1:15, then adding the mixture to buffers of different pH values under the same reaction conditions as those in step (1) of example 3. As shown in FIG. 5(C), when the pH values are 8.0 and 9.0, the relative enzyme activity reaches more than 90%, the optimum pH value for whole cell transformation is 8.0, and 0.2mol/L Na is selected in subsequent experiments₂HPO₄Citrate buffer (pH 8.0).

Measurement of optimum temperature: sublilis 168d1/pMA5 a-lacZ: mixing the subtiliss 168D1/pMA5a-araA at a cell number ratio of 1:15, and adding Na having a pH of 8.0₂HPO₄In a citric acid buffer, and setting reaction temperatures at 30, 40, 50, 60, 70 ℃, other reaction conditions were the same as in step (1) of example 3, and the relative enzyme activities of β -galactosidase and arabinose isomerase were measured. As shown in FIG. 5(D), when the temperature was 50 ℃, the relative enzyme activity was the highest, and the optimum temperature for whole-cell transformation was 50 ℃.

(2) Analysis of the concentration of the optimum metal ion for whole-cell transformation

Sublilis 168d1/pMA5 a-lacZ: B.Subtilis168D1/pMA5a-araA were mixed at a cell number ratio of 1:15, and Mn was added to each reaction system²⁺The optimum addition concentrations were measured at concentrations of 0, 0.5, 1.0, 3.0, 5.0, 7.0, 9.0 and 10.0mmol/L, and the other reaction conditions were the same as in step (1) of example 3. As shown in FIG. 5(E), Mn in the reaction system²⁺The optimum addition concentration of (2) is 3.0mmol/L with Mn²⁺The relative conversion rate is gradually reduced due to the increase of the added concentration, so that the optimum metal ion type for the whole cell conversion and the concentration of Mn are 3.0mmol/L²⁺。

(3) Analysis of optimal permeabilizing agent concentration for Whole cell transformation

Sublilis 168d1/pMA5 a-lacZ: the optimum addition concentrations of TritonX-100 in the reaction system were controlled to 0, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9 and 1.0% after mixing the subtiliss 168D1/pMA5a-araA at a cell number ratio of 1:15, and the other conditions were the same as in step (1) of example 3. As shown in FIG. 5(F), when the concentration of TritonX-100 added to the reaction system was 0.1%, the relative transformation rate gradually decreased with the increase in the concentration of TritonX-100 added, and therefore the optimum permeabilizing agent concentration for whole-cell transformation was 0.1% TritonX-100.

(4) Analysis of concentration of optimum bacterial suspension for whole cell transformation

In B.subtiliss 168D1/pMA5 a-lacZ: controlling the concentration OD of the bacterial suspension in the reaction system under the condition that the mixing ratio of the number of subtiliss 168D1/pMA5a-araA cells is 1:15₆₀₀After 20, 30, 40, 50, 60, 70, 50 ℃ reaction for 0, 12, 24, 48, 72 and 96h, 500. mu.l of the supernatant was collected by centrifugation and the reaction was terminated in a boiling water bath for 10min, and the other conditions were the same as in step (1) of example 3. As shown in FIG. 5(B), the concentration OD of the bacterial suspension₆₀₀When the concentration is 50, the highest production amount of tagatose is 26.96g/L, which is the concentration OD of the bacterial suspension₆₀₀The amount of tagatose produced was 1.7 at 20 hours, and therefore the optimum bacterial suspension concentration OD for whole cell transformation₆₀₀Is 50.

B. subtiliss 168d1/pMA5 a-lacZ: b. Subtilis168D1/pMA5a-araA bacterium was added at an optimum ratio of 1:15, and 3mmol/L Mn was added to 0.2M Na2HPO 4-citric acid (pH 8.0) at 50 ℃²⁺0.1 percent TritonX-100 and the concentration OD600 of the bacterial suspension is 50, the highest production amount of tagatose is the optimal whole-cell transformation condition.

Example 4: synthesis of tagatose

The whole cell transformation reaction system is expanded to be 1L, 100-500 g/L lactose is added under the optimized optimal condition, the pH of the reaction system is adjusted to be 8.0 all the time through 2mol/L HCl and NaOH, and 1mL of HPLC is sampled to detect the contents of a substrate, an intermediate product and a final product in the reaction system after 5, 10, 20, 40, 60 and 80 hours of reaction. As shown in FIG. 6, the tagatose yield was 30.05g/L and the maximum molar conversion rate was 57.23% when 100g/L of the substrate lactose was added, the tagatose yield was 96.76g/L and the molar conversion rate was 36.77% when the substrate loading was increased to 500 g/L.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for synthesizing tagatose from high-concentration lactose by catalyzing recombinant bacillus subtilis

<130> BAA210095A

<160> 5

<170> PatentIn version 3.3

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tatgacgtga ccttaacagg ttattctgtt gaatggcttc aggaggcagc ccgccacacg 360

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tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct 240

gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc 300

tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg 360

acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg 420

cgaattattt ttgatggcgt taactcggcg tttcatctgt ggtgcaacgg gcgctgggtc 480

ggttacggcc aggacagtcg tttgccgtct gaatttgacc tgagcgcatt tttacgcgcc 540

ggagaaaacc gcctcgcggt gatggtgctg cgctggagtg acggcagtta tctggaagat 600

caggatatgt ggcggatgag cggcattttc cgtgacgtct cgttgctgca taaaccgact 660

acacaaatca gcgatttcca tgttgccact cgctttaatg atgatttcag ccgcgctgta 720

ctggaggctg aagttcagat gtgcggcgag ttgcgtgact acctacgggt aacagtttct 780

ttatggcagg gtgaaacgca ggtcgccagc ggcaccgcgc ctttcggcgg tgaaattatc 840

gatgagcgtg gtggttatgc cgatcgcgtc acactacgtc tgaacgtcga aaacccgaaa 900

ctgtggagcg ccgaaatccc gaatctctat cgtgcggtgg ttgaactgca caccgccgac 960

ggcacgctga ttgaagcaga agcctgcgat gtcggtttcc gcgaggtgcg gattgaaaat 1020

ggtctgctgc tgctgaacgg caagccgttg ctgattcgag gcgttaaccg tcacgagcat 1080

catcctctgc atggtcaggt catggatgag cagacgatgg tgcaggatat cctgctgatg 1140

aagcagaaca actttaacgc cgtgcgctgt tcgcattatc cgaaccatcc gctgtggtac 1200

acgctgtgcg accgctacgg cctgtatgtg gtggatgaag ccaatattga aacccacggc 1260

atggtgccaa tgaatcgtct gaccgatgat ccgcgctggc taccggcgat gagcgaacgc 1320

gtaacgcgaa tggtgcagcg cgatcgtaat cacccgagtg tgatcatctg gtcgctgggg 1380

aatgaatcag gccacggcgc taatcacgac gcgctgtatc gctggatcaa atctgtcgat 1440

ccttcccgcc cggtgcagta tgaaggcggc ggagccgaca ccacggccac cgatattatt 1500

tgcccgatgt acgcgcgcgt ggatgaagac cagcccttcc cggctgtgcc gaaatggtcc 1560

atcaaaaaat ggctttcgct acctggagag acgcgcccgc tgatcctttg cgaatacgcc 1620

cacgcgatgg gtaacagtct tggcggtttc gctaaatact ggcaggcgtt tcgtcagtat 1680

ccccgtttac agggcggctt cgtctgggac tgggtggatc agtcgctgat taaatatgat 1740

gaaaacggca acccgtggtc ggcttacggc ggtgattttg gcgatacgcc gaacgatcgc 1800

cagttctgta tgaacggtct ggtctttgcc gaccgcacgc cgcatccagc gctgacggaa 1860

gcaaaacacc agcagcagtt tttccagttc cgtttatccg ggcaaaccat cgaagtgacc 1920

agcgaatacc tgttccgtca tagcgataac gagctcctgc actggatggt ggcgctggat 1980

ggtaagccgc tggcaagcgg tgaagtgcct ctggatgtcg ctccacaagg taaacagttg 2040

attgaactgc ctgaactacc gcagccggag agcgccgggc aactctggct cacagtacgc 2100

gtagtgcaac cgaacgcgac cgcatggtca gaagccgggc acatcagcgc ctggcagcag 2160

tggcgtctgg cggaaaacct cagtgtgacg ctccccgccg cgtcccacgc catcccgcat 2220

ctgaccacca gcgaaatgga tttttgcatc gagctgggta ataagcgttg gcaatttaac 2280

cgccagtcag gctttctttc acagatgtgg attggcgata aaaaacaact gctgacgccg 2340

ctgcgcgatc agttcacccg tgcaccgctg gataacgaca ttggcgtaag tgaagcgacc 2400

cgcattgacc ctaacgcctg ggtcgaacgc tggaaggcgg cgggccatta ccaggccgaa 2460

gcagcgttgt tgcagtgcac ggcagataca cttgctgatg cggtgctgat tacgaccgct 2520

cacgcgtggc agcatcaggg gaaaacctta tttatcagcc ggaaaaccta ccggattgat 2580

ggtagtggtc aaatggcgat taccgttgat gttgaagtgg cgagcgatac accgcatccg 2640

gcgcggattg gcctgaactg ccagctggcg caggtagcag agcgggtaaa ctggctcgga 2700

ttagggccgc aagaaaacta tcccgaccgc cttactgccg cctgttttga ccgctgggat 2760

ctgccattgt cagacatgta taccccgtac gtcttcccga gcgaaaacgg tctgcgctgc 2820

gggacgcgcg aattgaatta tggcccacac cagtggcgcg gcgacttcca gttcaacatc 2880

agccgctaca gtcaacagca actgatggaa accagccatc gccatctgct gcacgcggaa 2940

gaaggcacat ggctgaatat cgacggtttc catatgggga ttggtggcga cgactcctgg 3000

agcccgtcag tatcggcgga attccagctg agcgccggtc gctaccatta ccagttggtc 3060

tggtgtcaaa aatga 3075

<210> 3

<211> 1503

<212> DNA

<213> Artificial sequence

<400> 3

atgacgattt ttgataatta tgaagtgtgg tttgtaattg gcagccagca tctgtatggc 60

ccggaaaccc tgcgtcaggt cacccaacat gccgagcacg tcgttaatgc gctgaatacg 120

gaagcgaaac tgccctgcaa actggtgctg aaaccgctgg gcaccacgcc ggatgaaatc 180

accgctattt gccgtgacgc gaattacgac gatcgttgcg ctggtctggt ggtgtggctg 240

cacacctttt ccccggccaa aatgtggatc aacggcctga ccatgctcaa caaaccgttg 300

ctgcaattcc acacccagtt caacgcggcg ctgccgtggg atagcatcga tatggacttt 360

atgaacctga accagactgc acatggcggt cgcgagttcg gcttcattgg cgcgcgtatg 420

cgtcagcaac atgctgtcgt taccggtcac tggcaggata aacaagcaca tgagcgtatc 480

ggctcctgga tgcgtcaggc ggtctctaaa caggataccc gtcatctgaa agtctgccgt 540

tttggcgata acatgcgtga agtagcggtc accgatggcg ataaagttgc cgcacagatc 600

aagtttggtt tctccgtcaa tacctgggcg gttggcgatc tggtgcaggt ggtgaactcc 660

atcagcgatg gcgatgttaa cgcgctggtc gatgagtacg aaagctgcta caccatgacg 720

cctgcgacac aaatccacgg cgaaaaacga cagaacgtgc tggaagcggc gcgtattgag 780

ctggggatga agcgtttcct ggaacaaggt ggcttccacg cgttcaccac cacctttgaa 840

gatttgcacg gtctgaagca gcttcctggt ctggccgtac agcgtctgat gcagcagggc 900

tacggctttg cgggcgaagg cgactggaaa actgccgccc tgcttcgcat catgaaggtg 960

atgtcaaccg gtctgcaggg cggcacctcc tttatggagg actacactta ccacttcgaa 1020

aaagataatg acctggtgct cggctcccat atgctggaag tctgtccgtc gatcgccgta 1080

gaagagaaac cgatcctcga cgttcaacac ctcggtattg gcggtaaaga cgatcctgcc 1140

cgcctgatct tcaacactca aaccggtccg gccattgtcg ccagtctgat tgatctcggc 1200

gatcgttacc gtctgctggt taactgcatc gacactgtga aaacaccgca ctccctgccg 1260

aaactgccgg tggcgaatgc gctgtggaaa gcgcaaccgg atctgccaac tgcttccgaa 1320

gcgtggatcc tcgctggtgg cgcgcaccat accgttttca gccatgcgct gaacctcaac 1380

gatatgcgcc agttcgccga gatgcacgac attgaaatca cggtgattga taacgacacc 1440

cgcctgccag cgtttaaaga cgcgctgcgc tggaacgaag tgtattacgg atttcgtcgc 1500

atg 1503

<210> 4

<211> 759

<212> DNA

<213> Artificial sequence

<400> 4

gtgaatggac caataataat gactagagaa gaaagaatga agattgttca tgaaattaag 60

gaacgaatat tggataaata tggggatgat gttaaggcta ttggtgttta tggctctctt 120

ggtcgtcaga ctgatgggcc ctattcggat attgagatga tgtgtgtcat gtcaacagag 180

gaagcagagt tcagccatga atggacaacc ggtgagtgga aggtggaagt gaattttgat 240

agcgaagaga ttctactaga ttatgcatct caggtggaat cagattggcc gcttacacat 300

ggtcaatttt tctctatttt gccgatttat gattcaggtg gatacttaga gaaagtgtat 360

caaactgcta aatcggtaga agcccaaacg ttccacgatg cgatttgtgc ccttatcgta 420

gaagagctgt ttgaatatgc aggcaaatgg cgtaatattc gtgtgcaagg accgacaaca 480

tttctaccat ccttgactgt acaggtagca atggcaggtg ccatgttgat tggtctgcat 540

catcgcatct gttatacgac gagcgcttcg gtcttaactg aagcagttaa gcaatcagat 600

cttccttcag gttatgacca tctgtgccag ttcgtaatgt ctggtcaact ttccgactct 660

gagaaacttc tggaatcgct agagaatttc tggaatggga ttcaggagtg gacagaacga 720

cacggatata tagtggatgt gtcaaaacgc ataccattt 759

<210> 5

<211> 399

<212> DNA

<213> Artificial sequence

<400> 5

atgttacagt ctatcccggc attgccagtc ggggatatta aaaagagtat aggtttttat 60

tgcgataaac taggtttcac tttggttcac catgaagatg gattcgcagt tctaatgtgt 120

aatgaggttc ggattcatct atgggaggca agtgatgaag gctggcgctc tcgtagtaat 180

gattcaccgg tttgtacagg tgcggagtcg tttattgctg gtactgctag ttgccgcatt 240

gaagtagagg gaattgatga attatatcaa catattaagc ctttgggcat tttgcacccc 300

aatacatcat taaaagatca gtggtgggat gaacgagact ttgcagtaat tgatcccgac 360

aacaatttga ttagcttttt tcaacaaata aaaagctaa 399

Claims

1. A composition comprising a recombinant bacterium which expresses beta-galactosidase and a recombinant bacterium which expresses arabinose isomerase.

2. The composition of claim 1, wherein the recombinant bacterium expresses an alanine racemase gene alrA, and the nucleotide sequence is shown as SEQ ID No. 1.

3. The composition according to claim 1, wherein the nucleotide sequences of the beta-galactosidase and arabinose isomerase are shown as SEQ ID No.2 and SEQ ID No. 3.

4. The composition of any one of claims 1 to 3, wherein the recombinant bacterium is a Bacillus subtilis168 as a host and pMA5 as an expression vector.

5. The composition of claim 4, wherein the Bacillus subtilis B.subtilis168 has removed the alanine racemase gene alrA; the vector pMA5 removed the kanamycin and bleomycin resistance genes.

6. A method for synthesizing tagatose, which is characterized in that lactose is used as a substrate, the composition of claim 1 is added, and the reaction lasts for 70-90 hours.

7. The method of claim 6, wherein the method is carried out at a pH of 8.0 to 9.0 and a temperature of 45 to 55 ℃.

8. The method according to claim 6, wherein the reaction system of the method has a cell number ratio of the recombinant bacterium expressing the beta-galactosidase to the recombinant bacterium expressing the arabinose isomerase of 1: (15-20); the concentration of the bacterial suspension in the reaction system of the method is OD₆₀₀40 to 50.

9. The method according to claim 6, wherein 2-5 mmol/L of Mn is added to the reaction system²⁺And a permeabilizing agent 0.1% TritonX-100.

10. Use of the composition according to any one of claims 1 to 5 or the synthetic tagatose according to any one of claims 6 to 9 for the preparation of foods, pharmaceuticals, nutraceuticals or cosmetics.